Análisis del PEI: (Proyecto Educativo Institucional)

2.1.1.1 Strains

The organisms and strains used in this study are listed in Tab. 2.1-2.3.

Table 2.1: Staphylococcus aureus strains

Strain Properties Reference

Newman reference strain Duthie and Lorenz (1952) Newman-29 DsaePQRS::kan Mainieroet al.(2010) Newman-29, 33 complementation of Newman-29 Mainieroet al.(2010) Newmancoa Dcoa::tetK McDevittet al.(1992) NewmanvWbp emp * Demp::erm Johnsonet al.(2008) Newmaneap Deap::erm Hussainet al.(2002) Newmanica Dica::tet Kropecet al.(2005) CA-MRSA USA300 DsaePQRS::kan Diepet al. (2006) RN4220 restriction-deficient derivative of

S. aureus8325-4

Kreiswirthet al.(1983)

* Formerly denoted as Newman emp (Johnson et al., 2008). Sequencing analysis re- vealed that assumedly by phage transduction not only theempgene but also the neighbor- ing functional vWbp gene of strain Newman has been replaced by a truncated version of vWbp from strain RN4220. This was further confirmed by the fact that vWbp could be

detected in the supernatants of 3 h Newman wildtype cultures but not of this empmutant strain (Fig. 3.8, p. 54).

Table 2.2: Yersinia enterocolitica strains

Strain Properties Reference

WA(pYV) clinical isolate WA-314, serotype O:8 Heesemannet al.(1983) WA-C plasmid-cured derivative of WA(pYV) Heesemannet al.(1983) WA(pYV lcrD) T3SS-deficient by Tn5 insertion in lcrD

(pYV-515)

Heesemannet al.(1983) WA inv(pYV) Dinv::kan, WA-C-inv(pYV) Ruckdeschelet al.(1996) WA(pYV yadA) DyadA::kan; WA(pYVO8-A-0) Roggenkampet al.(1995) WA inv

(pYV yadA)

Dinv::spec,DyadA::kan This work

WA(pYV, pRFP) WA-314 harboring pLACrfp Oellerichet al.(2007) WA(pYV yadA,

pRFP)

WA(pYV yadA) harboring pLACrfp Oellerichet al.(2007) WA(pYV yopH) yopH mutant WA-C(pYV H) Trülzschet al.(2004) WA(pYV yopO) yopO mutant WA-C(pYV O) Trülzschet al.(2004) WA(pYV yopP) yopP mutant WA-C(pYV P) Trülzschet al.(2004) WA(pYV yopE) yopE mutant WA-C(pYV E) Trülzschet al.(2004) WA(pYV yopT) yopT mutant WA-C(pYV T) Trülzschet al.(2004) WA(pTTSS) WA-C harboring pTTSS which encodes the

T3SS apparatus of WA-314 but no yop

genes

Trülzschet al.(2003)

WA(pTTSS, pYopHSycH)

secretion of YopH Trülzschet al.(2003) WA(pTTSS,

pYopO)

secretion of YopO Trülzschet al.(2003) WA(pTTSS,

pYopP-bla)

secretion of YopP-bla MvPI*, H. Bouabe WA(pTTSS,

pYopEyen)

secretion of YopEyen Trülzschet al.(2003)

WA(pTTSS, pYopTyen)

secretion of YopTyen MvPI*, K. Trülzsch

WA(pTTSS, pYopEyps)

(Y. enterocoliticastrains, continued)

Strain Properties Reference

WA(pTTSS, pYopTyps)

secretion of YopTyps MvPI*, B. Czech

WA(pTTSS, pYopTyenC139S)

secretion of YopTyenC139S MvPI*, B. Czech

WA(pTTSS, pYopEypsR144A)

secretion of YopEypsR144A MvPI*, B. Czech

WA(pTTSS, pYopTypsC139S)

secretion of YopTypsC139S MvPI*, B. Czech and this

work WA(pTTSS,

pYopEyenK62R,K75Q)

secretion of YopEyenK62R,K75Q Hentschkeet al.(2007)

WA(pYV, pYopH- bla)

WA(pYV) harboring pYopH-bla: secre- taion of ayopH-blafustion

Cvetanovic (2011) WA(pYV lcrD,

pYopH-bla)

WA(pYV lcrD) harboring pYopH-bla: secretaion of ayopH-bla fustion

Cvetanovic (2011)

* MvPI = Strain collection Max von Pettenkofer-Institute

yopgenes for expresssion in WA(pTTSS) are derived fromY. enterocoliticaserotype O:8 or fromY. pseudotuberculosisserotype O:3, strain IP32953, and cloned in pACYC184. Chap- ter 3.1.3.1-3.1.3.3 deals only with Y. enterocolitica-derived yop genes, therefore YopEyen

and YopTyen and the respective plasmids are referred to as YopE and YopT only in this

section.

Table 2.3: Other microorganisms

Strain Properties Reference

Escherichia coli DH5↵ strain for plasmid modifi-

cations,recA1, hsdR17

Hanahan (1983)

Escherichia coli NU14 clinical UPEC isolate Hultgrenet al.(1986)

Yersinia pseudotuberculosis

YPIII(pIB1)

wildtype strain Logsdon and Mecsas (2003)

Yersinia pseudotuberculosis IP32953 wildtype strain Chainet al. (2004)

Salmonella entericaserovar Ty- phimurium SB824

(Other microorganisms, continued)

Strain Properties Reference

Entamoeba histolyticaHK 9 clinical isolate Geiman and Becker

(1953)

2.1.1.2 Plasmids

The plasmids used in this study are listed in Tab. 2.4.

Table 2.4: Plasmids

Plasmid Properties Reference

pSK236 Shuttle vector containing pUC19 cloned into the HindIII site of the S. aureus plasmid pC194

Gaskill and Khan (1988)

pEmp a 1.3 kb fragment containing the emp

gene from strain Newman was cloned into pSK236 (BamHI/PstI)

This work

pvWbp a 1.9 kb fragment containing the vWbp

gene from strain Newman was cloned into pSK236 (EcoRI/SalI)

This work

pvWbpEmp a 1.9 kb fragment containing thevWbpgene (EcoRI/BamHI) and a 1.3 kb fragment con- taining the emp gene (BamHI/PstI) from strain Newman were cloned into pSK236

This work

pBLUESCRIPT II KS(+)

cloning plasmid Fermentas, Walldorf pBS-YopTyps yopTsycTyps in pBLUESCRIPT II KS(+) This work

pBS-YopTypsC139S yopTsycTypsC139S in pBLUESCRIPT II

KS(+)

This work

pFPV-mCherry/2 mCherry expression vector Drecktrahet al.(2008) pM965-4 eGFP eGFP expression vector Stecheret al. (2004) pTurboYFP-B turboYFP expression vector Evrogen, Moscow, Rus-

sia MLC-GFP myosin light chain-GFP fusion for expres-

sion in eukaryotic cells

2.1.1.3 Oligonucleotides

The oligonucleotides used in this study are listed in Tab. 2.5.

Table 2.5: Oligonucleotides

Oligonucleotide Sequence

vWbp-f-EcoRI AAA GAATTC TAATGATATTAAATTAATCATATG vWbp-r-SalI AAA GTCGAC AATTATTGATATTGATAGTTAAGC vwb-r-BamHI AAA GGATCC AATTATTGATATTGATAGTTAAGC emp-f-BamHI AAA GGATCC AAGCTGAAAAACAATAAAAATGTT emp-r-PstI AAA CTGCAG TGTTACTTCCAACTTTCAAAGTAG invETfor CGCATTAGATTAATGCATCGTGAAAAATGCAGAGAG TCTATTTTATGAGAAGTGGCGGTTTTCATGGCTTG invETrev GGTCACGCTAAAGGTGCCAGTTTGCTGGGCCGCAA GATTGGTATTTAGCACATTATTTGCCGACTACCTTG pKD46betFOR CCTTTCCTGATAAGCAGAATG pKD46betREV AATCCAAGAGCTTTTACTGC IP32953 pYV 29282 XbaI

forward

AATCTAGATAACATCACTGTCCCCATAAG IP32953 pYV 30915 SalI

reverse

TATGTCGACAGATATTATTGTAACTATGTAGTG YopT C139S Yps Forward CTAGCGGTGTCAGCGAGGCTTTATGTG

YopT C139S Yps Reverse GTGCACATAAAGCCTCGCTGACACGCCTAG Pbs-f GCAAGGCGATTAAGTTGGGTAAC

Pbs-r GCGCGCAATTAACCCTCAD pACYC184-1356-forward CAGAGCAAGAGATTACGCGCAG pACYC184-2312-reverse AAAATACAAAACGCCCGA

‘invETfor’-‘invETrev’were designed by K. Trülzsch, ‘pKD46betFOR/REV’ were de- signed by E. Schriefer. ‘IP32953 pYV 29282 XbaI forward’-‘Pbs-r’ were designed by B. Czech.